Identification Of SCIRR10 Protein Function Sites And Other Related Research

Spinal cord injury and regeneration related gene No 10 (SCIRR10) is a gene in our effort of seeking genes responsive to spinal cord neuron injury. Preliminary studies revealed that the gene located in almost the entire central nervous system. The relationship between SCIRR10 and spinal cord injury was proved by in situ hybridization, RT-PCR and Western Blot. SCIRR10 mRNA and protein expression were significantly upgraded after the spinal cord was injured. It was found SCIRR10 had interaction with thyrotropin-releasing hormone receptor 2 (TRH-R2) and carboxypeptidase E (CPE) by yeast two-hybrid technology. The interaction between SCIRR10 and receptor TRH-R2 was proved by protein Pull-Down technique, and TRH-R2 was confirmed as a receptor of SCIRR10 fatherly by using of ligand - receptor interaction technique. SCIRR10 could activate MAPK-ERK and phospholipase C signal transduction pathways. Activities of SCIRR10 were blocked by PTX, U73122 or U0126. SCIRR10 protein could significantly promote the survival of primary cortical neurons in vitro. SCIRR10 was a powerful promotion of the cortex and spinal cord nerve tissue regeneration. The purpose of this paper is reveal the SCIRR10 protein site of binding with TRH-R2. On the other hand, the interaction of SCIRR10 and CPE was confirmed.This paper mostly includes the following three parts: Part I: The functional site of SCIRR10 protein which bind with TRH-R2 was identified. SCIRR10 protein sequence was simulated and analysised by computer. SCIRR10 protein contains three sites of function as follows:F₄₆Y₅₄Y₆₄; F80Y81Y87; F128Y132Y136. Truncated SCIRR10 protein which had one,two or three sites were tested. On the other hand, peptide of mutations were tested also. F and Y was replaced by L or F was replaced by L and Y was replaced by F in the mutations of SCIRR10 protein fragments.1. Three truncated SCIRR10 protein were detected by GST-pull down and western blot. It was confirmed that three different truncated protein of SCIRR10 could bind with TRH-R2 in vitro.2. Truncated SCIRR10 protein and mutations were detected by fluorescence resonance energy transfer. The date of image which were collected to deal with statistics methods. It was confirmed truncated SCIRR10 protein which had F₄₆Y₅₄Y₆₄ could produce phenomenon of fluorescence resonance energy transfer with TRH-R2 in vivo. On the contrary, other groups couldn't produce the phenomenon. The site of F₄₆Y₅₄Y₆₄ is important in the binding with TRH-R2. 3. The localization of truncated SCIRR10 protein fragments and TRH-R2 in cells were deteceted by two Colour Fluorescence labeling protein technology. TRH-R2 was translated to the COS-7 cells. Expression of TRH-R2 was observed after translated 24h. The cells was treated with supernatant which had truncated SCIRR10 protein fragments. Truncated SCIRR10 protein which had F₄₆Y₅₄Y₆₄ could observe green fluorescence overlaying on the red fluorescence at the same cell. The site of F₄₆Y₅₄Y₆₄, should be playing an crucial role in the interaction of SCIRR10and TRH-R2. Part II: TRH-R2 mediated signaling pathways which were simulated by SCIRR10 protein fragments were detected. Truncated SCIRR10 protein fragments and mutations were used to stimulated the PC12 cells, and detected changes of TRH-R2 mediated signaling pathway by western blot.1. Expression of TRH-R2 gene on the PC12 cells was confirmed by RT-PCR and western blot.2. The PC12 cells was treated with supernatant which had truncated SCIRR10 protein fragments and mutations. The change of ERK1/2 phosphorylation and Elk-1 phosphorylation was detected by western blot. Only truncated SCIRR10 protein which had F₄₆Y₅₄Y₆₄ could cause changes. F₄₆Y₅₄Y₆₄ of SCIRR10 should be the function section.Part III: The interaction of SCIRR10 and CPE was confirmed. In early experiments, Carboxypeptidase E(CPE) and thyrotropin releasing hormone receptor 2(TRH-R2) were screened for proteins interaction with SCIRR10 by yeast two-hybrid technique. CPE is an enzyme which participates in the process of biosynthesis of neurotransmitter. The interaction between CPE and SCIRR10 should be tested by further experiments.1. CPE gene was cloned from rat brain cDNA library by RT-PCR. CPE gene was cloned into the vector pcDNA3.1/myc-His. CPE gene was expressed successfully on the COS-7 cells.2. The interaction of SCIRR10 and CPE was detected in vivo by fluorescence energy transfer technology. FRET efficiency was collected by Sensitized Emmission method. The image date was deal with statistical method. The FRET efficiency of SCIRR10 and CPE group far more than the negative control group.3. The protein-protein interaction of SCIRR10 and CPE in intro was detected. The prokaryotic expression vector of CPE was constructed at the beginning. The combination of SCIRR10 and CPE in vitro was confirmed by pull-down method.4.The position of SCIRR10 and CPE were identified in the cells by immunofluorescence staining method. SCIRR10 protein could overlap with the Golgi body. SCIRR10 protein and CPE protein overlapped at the cells which were cotransfected with SCIRR10 and CPE.In summary, F₄₆Y₅₄Y₆₄ is the key binding site of SCIRR10 and THR-R2. The SCIRR10 protein fragments which contain F₄₆Y₅₄Y₆₄ can activate TRH-R2-mediated the signaling pathways. The protein-protein interaction of SCIRR10 and CPE has been furtherly validated in vivo and in vitro. SCIRR10 protein was processed in the Golgi at first. SCIRR10 can be combined with CPE then. CPE is an important role during the process of SCIRR10.